Workshop on analytical methods

Molecularly imprinted polymers for trace analysis
Benoit Guieysse Biotechnology Dept., Lund University
The analysis of trace contaminants almost always requires preliminary steps of sample concentration and purification. Concentration is often conducted by solid-phase extraction (SPE) using silica-based adsorbents, which at best select the contaminants based on their hydrophobic properties, and must therefore be completed with further purification of the extract. Instead, a simpler and more efficient sample preparation procedure should be based on the selective extraction of the contaminants in order to avoid interfering substances to bind to the adsorbent. Molecularly Imprinting Polymers (MIPs) are prepared by polymerizing monomers around a template that is then removed, leaving a specific binding site available. If the template is the molecule to be analyzed, or an analogue, the MIP synthesized will be suitable for the selective extraction of the target molecule by molecular recognition and binding to the specific site. This principle has been demonstrated for many trace contaminants and has found numerous applications for extraction (SPE phase), analysis (HPLC column packing) and detection (MIP ligand binding assays). As a typical example, 17β-estradiol (E1) was recovered by 100 ± 0.6% from a SPE column packed with 100 mg of MIP synthesized with this compound and percolated with 2 l of an aqueous solution of 2 µg E1/l. This was significantly higher than the recovery of 87.1 ± 2.3% and 77.3 ± 5.2% achieved with a commercial C18 extraction phase and a control NIP (non imprinted polymer synthesized under the same conditions but without adding the template), suggesting the higher efficiency of the MIP was due to presence of the specific binding sites. The better properties of the MIP were confirmed when E1 was supplied in the presence of fluoxetine hydrochloride (FH) and acenaphthene (Ac) added as interferences: E1 extraction by the MIP (95.5 ± 4.0%) did not decrease significantly (p < 0.05) whereas the NIP and C18 efficiencies dropped to 54.5 ± 9.4% and 76.0 ± 2%, respectively. The potential of MIP as extraction phase is also especially interesting for measuring the endocrine activity in environmental sample because this normally requires a concentration step. By using an endocrine disrupter as template, the MIP synthesized should permits to remove even unknown contaminants having the capacity to bind natural receptors (i.e. all endocrine disrupters). Thus, the knowledge of the nature of each contaminant is no longer required and, if a molecule is toxic because it can bind with a natural biological receptor, it will be removed by the synthetic receptor analogues. This was demonstrated when extracting a real wastewater analysis with the E1-MIP and NIP described above: endocrine activity (equivalent to the effect caused by 22 ng E1/l as measured by the recombinant yeast assay) was only significant in the MIP extract, showing that current protocol might underestimate the real endocrine activity in environmental samples.